Vol. 136, No. 2

JOURNAL OF BACTERIOLOGY, Nov. 1978, P. 597-606 0021-9193/78/0136-0597$02.00/0 Copyright ( 1978 American Society for Microbiology

Printed in U. S. A.

Carbon Monoxide Oxidation by Clostridium thermoaceticum and Clostridium formicoaceticum GABRIELE B. DIEKERT AND RUDOLF K. THAUER* Fachbereich Biologie-Mikrobiologie, Philipps- Universitat Marburg, D-3550 Marburg, Federal Republic of Germany Received for publication 4 May 1978

Cultures of Clostridium formicoaceticum and C. thermoaceticum growing on fructose and glucose, respectively, were shown to rapidly oxidize CO to CO2. Rates up to 0.4 timol min-' mg of wet cells-' were observed. Carbon monoxide oxidation by cell suspensions was found (i) to be dependent on pyruvate, (ii) to be inhibited by alkyl halides and arsenate, and (iii) to stimulate CO2 reduction to acetate. Cell extracts catalyzed the oxidation of carbon monoxide with methyl viologen at specific rates up to 10 ytmol min' mg of protein-' (35°C, pH 7.2). Nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate and ferredoxin from C. pasteurianum were ineffective as electron acceptors. The catalytic mechanism of carbon monoxide oxidation was "ping-pong," indicating that the enzyme catalyzing carbon monoxide oxidation can be present in an oxidized and a reduced form. The oxidized form was shown to react reversibly with cyanide, and the reduced form was shown to react reversibly with alkyl halides: cyanide inactivated the enzyme only in the absence of carbon monoxide, and alkyl halides inactivated it only in the presence of carbon monoxide. Extracts inactivated by alkyl halides were reactivated by photolysis. The findings are interpreted to indicate that carbon monoxide oxidation in the two bacteria is catalyzed by a corrinoid enzyme and that in vivo the reaction is coupled with the reduction of CO2 to acetate. Cultures of C. acidi-urici and C. cylindrosporum growing on hypoxanthine were found not to oxidize CO, indicating that clostridia mediating a corrinoid-independent total synthesis of acetate from CO2 do not possess a CO-oxidizing system.

Several anaerobic bacteria have been reported to oxidize carbon monoxide to C02: Clostridium pasteurianum (16, 37), C. welchii (33), Desulfovibrio desulfuricans (24, 45-47), a species of Rhodopseudomonas (20, 40), and all methanogenic bacteria tested (7, 21). Carbon monoxide oxidation is coupled with the reduction of CO2 to methane in methanogenic bacteria (7, 21) and with the reduction of protons to H2 in the Rhodopseudomonas species (40). The electron accepting reaction in the other anaerobic bacteria is not known. In this invesigation it is shown that C. formicoaceticum (1) and C. thermoaceticum (12) rapidly oxidized carbon monoxide to CO2 and that the reaction was coupled with the reduction of CO2 to acetate. The rates of carbon monoxide oxidation observed were more than 10 times higher than the highest rate so far reported for an anaerobic bacterium (7). The mechanism of carbon monoxide oxidation was investigated. MATERIALS AND METHODS Source of materials. CO (99.997%), N2 (99.996%), CO2 (99.998%), and He (99.996%) were obtained from

Messer Griesheim (Dusseldorf). Gas mixtures of CO and N2 were prepared in a gas burette (37). Coenzymes and enzymes were from Boehringer Mannheim (Mannheim). Ferredoxin from C. pasteurianum was prepared by the method of Schonheit et al. (26). Bovine serum albumin (92%), methyl viologen, and benzyl viologen were from Serva (Heidelberg). Triquat (1,1'-trimethylene-2,2'-dipyridyl dibromide) was a gift from B. G. White, Jealott's Hill Research Station, Bracknell, Berkshire, England. Methyl iodide (purum) was from Fluka, Buchs, Switzerland. Ethyl iodide, propyl iodide (for synthesis), and carbon tetrachloride (reagent grade) were from E. Merck (Darmstadt). Sodium [14C]carbonate (59 mCi/mmol), sodium [2'4C]acetate (57.7 mCi/mmol), and sodium ['4C]formate (60.7 mCi/mmol) were from Amersham Buchler (Braunschweig). 4CO was prepared from [14C]formic acid as described previously (16). C. formicoaceticum (ATCC 27076, DSM 92), C. thermoaceticum (DSM 521), L. acidi-urici (ATCC 7906, DSM 604), and C. cylindrosporum (ATCC 7905, DSM 605) were from the Deutsche Sammlung von Mikroorganismen

(DSM; Gottingen). Growth of the bacteria. C. thermoaceticum was anaerobically grown at 55°C under 100% CO2 on the medium described in the 1977 catalog of strains of the Deutsche Sammlung von Mikroorganismen. The me-

597

598

DIEKERT AND THAUER

dium (sterilized by autoclaving) contained, per liter of distilled water: glucose* H20 (sterilized separately), 20 g; yeast extract (Difco), 5 g; tryptone (Difco), 5 g; K2HPO4, 7 g; KH2PO4, 5.5 g; Na2MoO4 2H20, 2.4 mg; (NH4)2SO4, 0.5 g; MgSO4 7H20, 0.1 g; NaHSeO:, 0.15 mg; Fe(NH4)2(SO4)2-6H20, 40 mg; sodium thioglycolate, 0.25 g; resazurin, 2 mg; NaHCO:3 (sterilized seperately in a tightly closed bottle under an atmosphere of C02), 8 g. CO2 was bubbled through the medium until a pH of approximately 7 was reached. C. formicoaceticum was anaerobically grown at 35°C under N2 on the medium described in the 1977 catalog of strains of the Deutsche Sammlung von Mikroorganismen. The atmosphere contained some C02, which was evolved from bicarbonate added to the medium (see below). The medium (sterilized by autoclaving) contained, per liter of distilled water: fructose (sterilized separately), 5 g; yeast extract (Difco), 5 g; K2HPO4, 10 g; NaHCO3 (sterilized separately under C02), 10 g; trace element solution, 10 ml; vitamin Be, 1 mg; sodium thioglycolate, 0.75 g; nitrilotriacetic acid, 0.13 g; Fe(NH4)2(SO4)2-6H20, 40 mg; resazurin, 2 mg. The trace element solution contained, per liter of distilled water: ethylene-diaminetetraacetate, 0.5 g; ZnSO4.7H20, 10 mg; MnCl2.4H20, 3 mg; H3BO3, 30 mg; CoCl2.6H20, 20 mg; CuCl2-2H20, 1 mg; NiCl2.6H20, 2 mg; Na2MoO4-2H20, 3 mg. The final pH of the medium was 8. C. acidi-urici and C. cylindrosporum were grown at 35°C under N2 on the medium described by Wagner and Andreesen (42). The atmosphere contained some C02, which was evolved from bicarbonate added to the medium (see below). The medium (sterilized by autoclaving) contained, per liter of distilled water: hypoxanthine, 2 g; yeast extract (Difco), 1 g; K2HPO4, 4 g; MgSO4 7H20, 0.1 g; FeSO4 7H20, 5 mg; Na2SeO3 5H20, 30 Lg; Na2MoO4 2H20, 30 jig; Na2WO4 2H20, 30 [Lg; sodium thioglycolate, 0.75 g; resazurin, 2 mg; NaHCO: (sterilized separately under CO2), 2 g. For C. acidi-urici the medium was adjusted to a pH of 7.3; for C. cylindrosporum it was adjusted to a pH of 7.7. A 5% inoculum was used throughout. Growth of the bacteria was followed by measuring the absorbance at 578 nm. For mass culture the bacteria were grown in 14-liter carboys. The cells were harvested in the midto late-exponential phase of growth by centrifugation at 35,000 x g in a Sorvall RC-5 (DuPont Instruments) equipped with a KSB continuous-flow system. After centrifugation the culture medium was decanted, and the cell pellet was immediately frozen in liquid nitrogen. The steel tubes containing the cells were closed under N2 with a rubber stopper and stored at -70°C. Preparation of cell extracts. One gram (wet weight) of cells was suspended in 3 ml of 100 mM potassium phosphate buffer (pH 7.2) containing 2 mg of lysozyme, 0.2 mg of deoxyribonuclease, and 50 mM 2-mercaptoethanol. The suspension was incubated under N2 for 30 min at 35°C. More than 95% of the cells were lysed under these conditions. The lysate was centrifuged at 35,000 x g for 15 min. The supernatant containing the carbon monoxide-oxidizing activity had a protein concentration of approximately 30 mg/ml. Protein was determined by the biuret method (34). When stored at 4°C under strictly anaerobic condi-

J. BACTERIOL.

tions, between 10% (C. thermoaceticum) and 50% (C. formicoaceticum) of the CO-oxidizing activity was lost within 24 h. Freezing to -20°C completely inactivated the enzyme. For the inactivation experiments the lysate was diluted 20-fold with 100 mM potassium phosphate buffer containing 25 mg of bovine serum albumin per ml and 50 mM 2-mercaptoethanol. Reactivation by photolysis was more complete under these conditions. Assay conditions for carbon monoxide oxidation by growing cultures. Carbon monoxide oxidation experiments with growing cultures were conducted under strictly anaerobic conditions in 28-ml aluminum seal tubes (Bellco Glass, Inc.). The tubes were filled with 6.6 ml of medium under either N2 or CO2 and then fitted with a gray butyl rubber stopper and secured with aluminum serum bottle seals (Wheaton Scientific). Carbon monoxide was then added with a gas-tight syringe. After inoculation with 0.4 ml of a growing culture, the tubes were incubated horizontally at either 55 or 35°C at 120 oscillations/min. At the times indicated gas and culture samples were removed with syringes and analyzed for CO and fermentation products. For the gas samples a gas-tight syringe with a Teflon pressure lock device (Mininert syringe valve, Supelco Inc.) was used. Growth of C. formicoaceticum, C. acidi-urici, and C. cylindrosporum was followed by measuring directly the increase in absorbance at 578 nm (ID of the tubes, ca. 1.2 cm); tubes filled with medium were used as a blank. For growth determination of C. thermoaceticum, 50-tld culture samples were removed and diluted 20-fold with 100 mM phosphate buffer (pH 7.2) before measuring the increase in absorbance at 578 nm. When the cells were grown in the presence of "4CO, the cultures were acidified at the end of growth by injection of 0.5 ml of 1 M HCl04. After equilibration of the gas with the liquid phase, gas samples were withdrawn and analyzed for "CO2. Assay conditions for carbon monoxide oxidation by cell suspensions. Carbon monoxide oxidation experiments with cell suspensions of C. formicoaceticum were conducted under strictly anaerobic conditions in 35-ml serum bottles (Wheaton Scientific). The bottles were filled under N2 with 4 ml of a medium containing 250 timol of potassium phosphate buffer (pH 7.7), 250 ,umol of 2-mercaptoethanol, 40 ytg of resazurin, and, where indicated, 500 ,umol of sodium pyruvate, 25 ,imol of carbon tetrachloride, 25 timol of propyl iodide, 120 ymol of arsenate, or 1 tlmol of 14CO2. After the serum bottles were closed with a gray butyl rubber stopper, CO was added with a gas-tight syringe. This was followed by injection of 10 1l of an anaerobically prepared sodium dithionite solution (50 mg in 5 ml of 0.5 M potassium phosphate, pH 7.2) with a microliter syringe. Then the bottles were preincubated at 35°C at 100 oscillations/min to remove remaining traces of 02 from the gas phase. The reaction was started by the addition of 1 ml of concentrated cell suspension, which was prepared by suspension of 25 to 500 mg of frozen cells in I ml of 50 mM potassium phosphate buffer (pH 7.7) containing 50 mM 2-mercaptoethanol. At the times indicated gas and suspension samples were removed and analyzed for carbon monoxide, pyruvate, and fermentation products. For the gas sample a gas-tight syringe with a Teflon pres-

VOI,. 136, 1978

CO OXIDATION BY CLOSTRIDIA

599

sure lock device (Mininert syringe valve) was used. At model 270 with a flame ionization detector equipped the end of the experiment 0.5 ml of 1 M HCl04 was with a glass column (1.8 m by 0.64 cm) filled with injected to acidify the cell suspension. After equilibra- Firebrick 60/80, 10% NPGS, and 2% H3PO4 (Werner tion of the gas and liquid phases, gas samples were Gunther Analysentechnik, Dusseldorf) and heated at withdrawn and analyzed for CO2. 150°C. N2 was used as carrier gas at a flow rate of 30 Assay conditions for carbon monoxide oxida- ml/min. Samples of 0.1 to 0.5 Ml (acidified to pH 2 with tion by cell extracts. The carbon monoxide-oxidizing HCl) were injected. Acetate was quantitated relating activity was measured by photometric determination the peak areas to a standard curve. of the reduction of methyl viologen with carbon mon['4C]acetate was counted by liquid scintillation after oxide at 578 nm (E578 = 9.7 mM cm-') (7). One unit of separation from other labeled compounds. One-millienzyme activity is defined as 1 Mmol of CO oxidized liter samples were acidified to pH 4 with HCl04 and per min or 2 ,umol of methyl viologen reduced by CO then extracted with ether for 24 h, using a Kutscherper min at 35°C and pH 7.2. The assays were con- Steudel apparatus. ['4C]acetate was recovered in the ducted in 1-ml cuvettes with ground-glass fittings ether phase. After extraction of the ether phase with closed with gray butyl rubber stoppers. The cuvettes water, ['4C]acetate was separated from other labeled were filled with 1 ml of 50 mM potassium phosphate compounds by isoionic ion-exchange chromatography buffer containing 5 mM methyl viologen or the con- (39). '4C was quantitated in 15 ml of Bray scintillator centration indicated. Anaerobic conditions were ob- (4), using ['4C]toluene as an internal standard. tained by alternate evacuation and gassing of the Determination of formate. Formate was meacuvettes with carbon monoxide or a CO/N2 mixture sured by the method described by Lang and Lang (22). while shaking and then addition of enough dithionite Samples, 100 Ml, containing 0.5 to 2.0 tImol of formate (5 to 50MM) to turn the test solution slightly blue. The were mixed with 0.2 ml of solution A, 10 ,ul of solution cuvettes were then warmed to the test temperature, X, and 0.7 ml of 100% acetic anhydride and then and the gas and liquid phases were equilibrated by incubated at 50°C for 30 min with occasional rapid vigorous shaking. The reaction was started by inject- mixing. A red color developed which was measured ing cell extract. The carbon monoxide concentration photometrically at 515 nm. Solution A was prepared in the aqueous solution at a given temperature was by dissolving 0.5 g of citric acid.lH20 and 10 g of calculated from solubility data (32). acetamide in 100 ml of isopropanol; solution B was Determination of CO. Carbon monoxide was mea- prepared by dissolving 30 g of sodium acetate in 100 sured gas chromatographically (7) on a Varian Aero- ml of water. graph model 1400 with thermal conductivity detection. Other determinations. H2 and CH4 were meaThe operating conditions were as follows: stainless- sured gas chromatographically as described previously steel column, 2.2 m by 0.32 cm, filled with Molecular (7). Ethanol, malate, lactate, and succinate were deSieve 5 A, 18 to 50 mesh, from E. Merck (Darmstadt); termined enzymatically. Glucose and fructose were helium carrier gas, 5 atm, 60 ml/min; column temper- assayed by the phenol method (19). ature, 100°C; detector temperature, 135°C; injection RESULTS port temperature, 45°C; filament current, 200 mA. Two-tenths-milliliter samples were injected. Carbon Carbon monoxide oxidation by growing monoxide was quantitated relating the peak heights to a standard curve. The carbon monoxide concentration cultures. C. formicoaceticum was grown on a in aqueous solution at a given temperature was cal- bicarbonate-buffered fructose medium and C. thermoaceticum was grown on a bicarbonateculated from solubility data (32). Determination of CO2. '2CO2 was measured gas buffered glucose medium, both in the presence chromatographically (7) on a Varian Aerograph model of carbon monoxide. The cultures rapidly re1400 with thermal conductivity detection. The operat- moved carbon monoxide from the gas phase ing conditions were as follows: stainless-steel column, (Fig. 1A and B). When 14CO was added most of 1.3 m by 0.32 cm, filled with Carbosieve B, 120/140 the radioactivity was recovered in C02, indicatmesh, from Supelco Inc.; helium carrier gas, 5 atm, 60 ing that carbon monoxide was oxidized to CO2. ml/min; column temperature, 100°C; detector temperBecause CO2 was reduced to acetate by the ature, 135°C; injection port temperature, 45°C; filament current, 200 mA. Two-tenths-milliliter samples growing bacteria, some radioactivity was also were injected. CO2 was quantitated relating the peak found in acetate. The portion of 14C recovered in heights to a standard curve. The CO2 concentration in acetate was small, however, because the CO2 aqueous solution at a given temperature was calcu- pool in the culture was large (>500 /Lmol) comlated from solubility data (32). pared to the amount of carbon monoxide oxi'4C02 was measured by liquid scintillation counting. dized (-50 Mmol). The rates of carbon monoxide Gas samples, 0.5 ml, were injected into a scfntillation oxidation were of the same order as the rate of vial that was closed with a rubber stopper. The vial contained 0.5 ml of 0.2 M KOH to absorb CO2. After fructose fermentation and glucose fermentation 6 h of shaking, 15 ml of Bray solution (4) was added, by the two bacteria (Fig. 1). Acetate and CO2 and the radioactivity was measured, using ['4C]toluene were the major fermentation products found as an internal standard. Under these conditions all of when the bacteria were grown in both the abthe CO2 was absorbed in the KOH. sence and the presence of carbon monoxide. H2 Determination of acetate. ['2C]acetate was mea- and CH4 or other reduced organic compounds sured gas chromatographically on a Varian Aerograph were not formed in significant amounts.

600

DIEKERT AND THAUER

J1. BACTFRIOL.

0

0

in

a co '-

c 0

c

2.' 4, U

0

Time (h)

c -C 0

0

1.4

.

Ch

u)

c4

c

.1:1

c

0

(D

0.!

C)

-

i

0

IU

Time(h)

FIG. 1. Carbon monoxide oxidation by cultures of C. thermoaceticum (A) and C. formicoaceticum (B) growing on glucose and fructose, respectively. Cultures, 7 ml, were continuously equilibrated with a 21ml gas phase containing 5% carbon monoxide initially. A change in absorbancy at 578 nm (AA57+) of 1 corresponds to a cell concentration of approximately 1 mg of wet cells ml-'.

At a concentration of 5% in the gas phase, CO oxidized by C. formicoaceticum at a specific rate of 0.4 limol min-' mg of wet cells-' and by C. thermoaceticum at a specific rate of 0.2 ymol min-' mg of wet cells-' (Fig. 1A and B). The specific rate of carbon monoxide oxidation declined with decreasing CO concentration. Growth was not affected by carbon monoxide up to a concentration of 5%. At higher concentrations, however, the lag phase was increasingly prolonged. Twenty percent CO added to an exponentially growing culture did not decrease the growth rate. A significant inhibition of carbo hydrate utilization by CO was not observed. C. acidi-urici and C. cylindrosporum were was

grown on a bicarbonate-buffered hypoxanthine medium at 35°C. During growth on hypoxanthine the bacteria are known to synthesize acetate from CO2 (31). The cultures of the two bacteria did not oxidize carbon monoxide under the experimental conditions used at a concentration of either 0.2 or 5% in the gas phase; the concentration of carbon monoxide remained essentially constant from the beginning to the end of growth. Carbon monoxide oxidation by cell suspensions. Cell suspensions of C. formicoaceticum in phosphate buffer oxidized carbon monoxide only very slowly (Fig. 2). Reduced products formed under these conditions were formate and acetate. Per mole of CO oxidized, approximately 0.5 mol of formate, 0.25 mol of C02, and 0.1 mol of acetate were found. When pyruvate was added to the system, the rate of carbon monoxide oxidation was increased more than 10fold (Fig. 2). The pyruvate added was fermented to acetate and C02; approximately 4 mol of pyruvate was consumed per mol of carbon monoxide oxidized. In the absence of carbon monoxide 4 mol of pyruvate was fermented to 5 mol of acetate and 2 mol of CO2. In the presence of carbon monoxide the same products were found; the amount of CO2 formed increased from 2 to nearly 2.7 mol; the amount of acetate formed also increased. The increase was, however, too small to be demonstrated unambiguously. An exact stoichiometry was difficult to obtain since -

Pyruvcite

+

Pyruvate

0

\

20 Ln

15

E

-

01 C

C 0

0

Or.< 0

2

4

6

8

Time( h) FIG. 2. Carbon monoxide oxidation by cell suspensions of C. formicoaceticum in the absence and presence of pyruvate (100 mM). Cell suspensions, 5 ml (5 mg of uet cells ml '), were continuously equilibrated with a 30-ml gas phase containing 20%( carbon mon-

oxide initially.

CO OXIDATION BY CLOSTRIDIA

VOL. 136, 1978

both acetate and CO2 were formed by the cells from endogenous sources in the absence of both carbon monoxide and pyruvate. Other products, including H2, CH4, lactate, malate, succinate, and ethanol, were sought but were not found in significant amounts. The oxidation of carbon monoxide by cell suspensions was almost completely inhibited by 5 mM carbon tetrachloride (Fig. 3). Propyl iodide (5 mM) and arsenate (25 mM) inhibited the reaction by more than 50%. Incubations with arsenate were done in morpholinopropane sulfonate (MOPS) buffer rather than in phosphate buffer. However, no attempts were made to deplete the amount of phosphate present in the cells. Cell suspensions of C. formicoaceticum reduced 14CO2 to ['4C]acetate in the presence of pyruvate. The rate of ['4C]acetate formation increased by 50% when 5% carbon monoxide was present in the gas phase. Lower concentrations were also stimulatory. Half-maximal stimulation was observed at a carbon monoxide concentration of 0.1%. Carbon monoxide oxidation by cell extracts. Cell extracts of C. formicoaceticum and C. thermoaceticum were found to catalyze the reduction of methyl viologen with carbon monioxide. CO was oxidized to CO2 as indicated by the finding that exactly 2 mol of methyl viologen was reduced per mol of carbon monoxide added to the cuvettes. At pH 7.2, a temperature of 0

(n) -c

.C U)

C)

15F

4)

a) C

c

10F

0

a

4)

5

u

C

0

(8U 0

0

1

2

3

4

5

Time (h) FIG. 3. Effect of carbon tetrachloride (5 mM) on carbon monoxide oxidation by cell suspensions of C. formicoaceticum. Cell suspensions, 5 ml (50 mg of wet cells ml-'; 100 mMpyruvate), were continuously equilibrated with a 30-ml gas phase containing 20%o carbon monoxide initially.

601

35°C, a methyl viologen concentration of 5 mM, and a carbon monoxide concentration in the gas phase of 100%, specific rates of carbon monoxide oxidation of 10 and 6 ymol min-' mg of protein-', respectively, were observed. The enzyme also catalyzed the reduction of benzyl viologen, triquat, and methylene blue. Nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, and ferredoxin from C. pasteurianum were ineffective as electron acceptors. The enzyme was found in the soluble cell fraction. It was sensitive to dilution in water but could, however, be stabilized by high ionic strength (>0.5 M potassium phosphate) or by the addition of bovine serum albumin (>20 mg/ml). Increase in ionic strength or the addition of bovine serum albumin did not, however, improve the stability of the enzyme to storage at +4 or -20°C. Exposure of the extract to air resulted in a complete and irreversible loss of the capacity to oxidize carbon monoxide. The reduction of methyl viologen with carbon monoxide was linear with time and protein in the range measurable photometrically. The dependence of the rate on the substrate concentration followed simple Michaelis-Menten kinetics: plots of l/v versus 1/S were linear. The kinetic constants given in Table 1 were determined at 35°C and pH 7.2. Vmalx for the enzyme of C. thermoaceticum at 55°C was 18 ,imol min-' mg of protein-'. The rate of methyl viologen reduction with CO increased with increasing pH [pH 5.5 to 6.5, sodium succinate/HCl; pH 6.5 to 7.5, K2HPO4/KH2PO4; pH 7.5 to 8.5, tris(hydroxymethyl)aminomethane-hydrochloride; pH 8.5 to 10.5, glycine/KOH] without reaching a plateau. A pH optimum was not observed. At pH 9 the rates were approximately 2.8 times higher than at pH 7.2; at pH 10.5 the rates were approximately 5 times higher than at pH 7.2. TABLE 1. Kinetics of the carbon monoxideoxidizing activity in cell extracts of C. formicoaceticum and C. thermoaceticum5 V (apparv ent) [MV]0.5 v l[CO]o5 Organism n(M) (mM) (U/mg of protein) C. formicoaceti2.2 3 15 cum C. thermoaceti150 1.3 7 cum Conditions: pH 7.2; temperature, 35°C; [S]0.5 V for methyl viologen (MV) determined at a CO concentration in the gas phase of 100% (= 0.8 mM); [S]0.5 v for CO determined at a methyl viologen concentration of 50 mM; V (apparent) determined at a CO concentration of 100% in the gas phase and a methyl viologen concentration of 50 mM. "

602

DIEKERT ANI) THAUER

J. BACTERIOL.

The apparent Km values for carbon monoxide and methyl viologen of the enzyme of C. thermoaceticum were high enough (Table 1) to allow a kinetic analysis of the catalytic mechanism. The concentrations of CO and methyl viologen were varied at different fixed levels of the other substrate, and initial velocities were determined. Double-reciprocal plots were parallel (Fig. 4), indicating that the catalytic mechanism of carbon monoxide oxidation is "ping-pong" (6).

When a cell extract of C. formicoaceticum was incubated at 35°C in the dark with 0.5 mM methyl iodide in the presence of carbon monoxide, a rapid decrease in carbon monoxide-oxidizing activity was observed (Fig. 5). The activity of the methyl iodide-inactivated enzyme could be restored by exposure of the extract at 0°C under an N2 gas phase to the light of a projection lamp. No reactivation was found in the dark. The enzyme of C. thermoaceticum, too, was inactivated by methyl iodide (2.5 mM) in the presence of CO and could be reactivated by photolysis under the described conditions. Similar effects on the enzyme of the two bacteria were observed with ethyl iodide, propyl iodide, and carbon tetrachloride. However, carbon tetrachloride-inactivated extracts could not be reactivated by photolysis. Inactivation of the enzyme catalyzing carbon monoxide oxidation was greatly enhanced by CO. In the absence of carbon monoxide the rate of inactivation was only slow (Table 2). Incubation of the extracts with cyanide in the absence of carbon monoxide resulted in a re100

2 0

1

[Methyl viologenj

(mM-')

o

l,

o 0

E

5.

.0

'7

60 -

C

3

B

S [Methyl

viologerl(mM)

~~~~~~0 C

4.1

0

20C

.15 D~3 ;0

O

o

2 2Y

/~~~~~10.

I

0

~~~~Dark control

0J 0 30 60 Time (min)

.~~~~~~~~~~~~~~~~~~~ 5 I

10

1S

20

25

(mM-')

FIG. 4. Kinetics of methyl viologen reduction with carbon monoxide in cell extracts of C. thermoaceticum at different fixed levels of carbon monoxide (A) or methyl viologen (B). Conditions: pH 7.2; tempera-

ture, 250C; enzyme, 12 pg'of protein of cell extract; volume, 1 ml. One unit = 1 ,zmol of CO oxidized min-' = 2 Mimol of methyl viologen reduced min-1.

FIG. 5. Reversible inactivation of the carbon monoxide-oxidizing activity with methyl iodide in cell extract of C. formicoaceticum. Two-milliliter diluted cell extracts (pH 7.7) were incubated at 350C in the dark with 0.5 mM methyl iodide under 100% carbon monoxide as the gas phase. The liquid and gas

phases were continuously equilibrated by shaking. After 10 min the temperature was lowered to 0°C and the gas phase was changed to 100%/ N2. Except for the control the reaction mixture was then exposed at 0°C to the light of a projection lamp (250 W/24 V), projector type Prado from E. Leitz. The distance between the test tube and the lamp was 7 cm. At the times indicated 5-jil samples were withdrawn and assayed for carbon monoxide-oxidizing activity.

VOL. 136, 1978

CO OXIDATION BY CLOSTRII)IA

TABLE 2. Inactivation of the carbon monoxideoxidizing activity in cell extracts of C. formicoaceticum and C. thermoaceticum by cyanide and by methyl iodidea Organism

Activity (%)h after 30-min inactivation by: CN

CH I1

C. formicoaceticum -Co 19.3 87.1 +CO 100 33.8 C. thermoaceticum -CO 29.6 95.4 +CO 100 35.1 a For assay conditions see Fig. 5 and 6. Cyanide concentration, 5 AM for C. formicoaceticum and 40 jIM for C. thermoaceticum; methyl iodide concentration, 0.5 mM for C. formicoaceticum and 2.5 mM for C. thermoaceticum. b Percentage of initial activity.

versible loss of the carbon monoxide-oxidizing activity. In the presence of carbon monoxide no inactivation was observed (Table 2). Carbon monoxide not only prevented but also reversed the inactivation by cyanide. When inactivated extracts were incubated with carbon monoxide, 100% of the initial activity reappeared within a few minutes (Fig. 6). Azide, fluoride, and ethylenediamine tetraacetate at concentrations of 10 mM neither inactivated nor inhibited the carbon monoxide-

603

in both the absence and the presence of carbon monoxide. Other products were looked for but not found in significant amounts, indicating that carbon monoxide oxidation was coupled with the reduction of CO2 to acetate. This was substantiated by experiments with cell suspensions of C. formicoaceticum. The following observations were made. (i) Carbon monoxide oxidation was stimulated more than 10-fold by pyruvate (Fig. 2). CO2 reduction to acetate in C. formicoaceticum and C. thermoaceticum requires pyruvate, since the carboxyl of acetate is derived from the carboxyl of pyruvate by a mechanism involving reductive transcarboxylation (30). Pyruvate may also be required as an additional electron source for the reduction of CO2 to acetate. Any oxidation reaction coupled with the reduction of CO2 to acetate must therefore be dependent on pyruvate. The slow rate of carbon monoxide oxidation observed in the absence of pyruvate can be explained by the formation of pyruvate from endogenous sources (30). Considerable amounts of CO2 and acetate were formed by the cells in the absence of both carbon monoxide and pyruvate.

(ii) Carbon monoxide oxidation significantly increased the rate of CO2 reduction to acetate in 100

oxidizing activity. DISCUSSION C. formicoaceticum and C. thermoaceticum are obligate anaerobic bacteria that use CO2 as electron acceptor for catabolic oxidation reactions. The reducing equivalents liberated in the oxidative part of metabolism are transferred to C02, which thereby is reduced to acetate; formate, 10- formyltetrahydrofolate, methenyltetrahydrofolate, methylenetetrahydrofolate, methyltetrahydrofolate, and an enzyme-bound methylcorrinoid have been shown to be intermediates. By a mechanism not yet fully understood, the methylcorrinoid is carboxylated to yield acetate. This step is dependent on pyruvate, and evidence has been presented that the carboxyl of acetate is derived from the carboxyl of the a-ketoacid by a mechanism involving reductive transcarboxylation. The evidence for this pathway of synthesis of acetate from CO2 has been reviewed (23, 38). In this investigation it was shown that C. formicoaceticum and C. thermoaceticum growing on hexoses rapidly oxidize carbon monoxide to CO2. Acetate was the only major fermentation product formed when the bacteria were growing

80 60-~~~~~~ 60~~ C

V)

g1o ~~~~~Control

o

0. 0

4

8

12

I 16

Time (min) FIG. 6. Reversible inactivation of the carbon monoxide-oxidizing activity with cyanide in cell extracts of C. formicoaceticum. Two-milliliter diluted cell extracts (pH 7.2) uwere incubated at 0°C with 2 PiM potassium cyanide under N2. The gas and liquid phases uwere continuously equilibrated by shaking. After 6 min, except for the control, the gas phase was changed to 100%, CO. At the times indicated 5-jil samples were withdrawn and assayed for carbon monoxide-oxidizing activity.

604

DIEKERT AND THAUER

the presence of pyruvate. This finding shows directly that carbon monoxide supplied electrons for the reduction of C02 to acetate. (iii) Carbon monoxide oxidation was effectively inhibited by carbon tetrachloride and propyl iodide. Acetate formation from C02 is inhibited by these alkyl halides, which exert their effect by alkylation of the corrinoid enzyme catalyzing the reduction of methyltetrahydrofolate to acetate (17). (iv) Carbon monoxide oxidation was inhibited by arsenate. Arsenate is known to inhibit ATP synthesis in clostridia. Acetate formation from C02 is dependent on ATP (23, 38), which is required for the activation of formate to formyltetrahydrofolate. Carbon monoxide oxidation by cell suspensions of C. formicoaceticum was accompanied by a rapid fermentation of pyruvate to acetate and CO2. At least 4 mol of pyruvate was fermented per mol of carbon monoxide oxidized: 4 pyruvate + 1 CO + 2.5 H20 -f 5.25 acetate + 2.5 C02 + 1.25 H+

Intermediate equations are: 4 pyruvate- + 2 H20 -

5 acetate + 2 C02 + 1H+

1 CO + 1 H20-- 1 C02 + 2 H+ + 2 e

0.5 C0 + 2 H+ + 2 e -*

0.25 acetate + 0.25 H+

Thus, at most two of the eight electrons required for CO, reduction to acetate were supplied by CO. However, other conditions may be required to obtain ratios higher than 2:8. Carbon monoxide was cometabolized by C. formicoaceticum and C. thermoaceticum rather than used as the sole energy source; carbon monoxide oxidation was dependent on the fermentation of carbohydrates or pyruvate. In this respect carbon monoxide oxidation in the two clostridia is similar to that in methane-oxidizing bacteria, which oxidize carbon monoxide only when metabolizing other substrates (9-1 1). Coupling of carbon monoxide oxidation with acetate formation from C02 could occur via the enzymatic reduction by CO of an electron carrier involved in C02 reduction to acetate (18). An alternate mechanism would be that CO directly reduces the prosthetic group of one of the enzymes participating in the reduction of C02 to acetate. There are some indications that the oxidation of CO is catalyzed by a corrinoid en-

J. BACTERIOL.

zyme, as is the reduction of methyltetrahydrofolate to acetate, which would be in support of the latter coupling mechanism. The evidence for this is discussed below. The catalytic mechanism of carbon monoxide oxidation was found to be "ping-pong." A prosthetic group of the enzyme is thus reduced by carbon monoxide and subsequently reoxidized by the electron acceptor (6). The fact that the enzyme reacts with carbon monoxide indicates that the prosthetic group contains a transition metal. The finding that methyl viologen (E', = -440 mV) and triquat (E', = -548 mV) can be used as electron acceptors leads to the conclusion that the redox potential of the electronaccepting group must be more negative than -400 mV. The reduced form of the enzyme was found to specifically react with alkyl halides, and the oxidized form was found to react with cyanide; the alkyl halide-inactivated enzyme was reactivated by photolysis. The enzyme was not inhibited by azide, fluoride, cyanate, and ethylenediaminetetraacetate. These properties can be best explained if it is assumed that the prosthetic group of the carbon monoxide-oxidizing activity is a vitamin B12 compound. Vitamin B12 can exist in an oxidized form [cob(III)alamin], in a reduced form [cob(II)alamin], and in a superreduced form [cob(I)alamin] and has a reactive 6 coordination position (see 14). Carbon monoxide is known to react with cob(II)alamin, which is reduced to cob(I)alamin by carbon monoxide (27, 28; also see 13, 15). Cob(I)alamin can be reoxidized by viologen dyes and methylene blue (29). The redox potential of the cob(II)alamin/ cob(I)alamin couple is lower than -420 mV, as indicated by the finding that cob(I)alamin can reduce protons at pH 7 to H2 (3). Cob(I)alamin can specifically be alkylated by alkyl halides; the alkylcob(I)alamin formed is photolysed to cob(II)alamin and an alkyl radical (14). Corrinoid enzymes involved in methyl transfer reactions are inactivated by alkylation of the cobalt atom with alkyl halides under reducing conditions in the dark. These enzymes are reactivated by photolysis (35, 36, 43, 44). Cob(III)alamin slowly forms a very stable cyanocob(III)alamin complex (11). Vitamin B12 does not form stable complexes with other ligands to transition metals such as azide, fluoride, and cyanate (11). The properties observed for the carbon monoxide-oxidizing activity are thus in accord with the involvement of a corrinoid compound in carbon monoxide oxidation; however, they do not prove it. Final evidence will have to await

VOL. 13fi, 1978

CO OXII)ATION BY CLOSTRIDIA

purification of the system. A corrinoid enzyme has been shown to catalyze the last step in acetate formation from CO2 in C. thermoaceticum and C. formicoaceuicum (23). This catabolic enzyme can be inactivated by alkyl halides and reactivated by photolysis and also reacts with cyanide (17). The high activity of CO oxidation in C. formicoaceticum and C. thermoaceticum and the finding that the carbon monoxide-oxidizing activity of the two bacteria is reversibly inactivated by alkyl halides and cyanide indicate that acetate formation from methyltetrahydrofolate and carbon monoxide oxidation may be catalyzed by the same enzyme system. This would be in anology to methane-oxidizing bacteria, in which carbon monoxide oxidation is catalyzed by methane monooxygenase (9-11). The conclusion is speculative and remains to be proven. Acetobacterium woodii can grow on hydrogen and CO, as sole energy source; CO2 is reduced to acetate (2, 25). Cell extracts of this organism have been observed to catalyze the reduction of methyl viologen with carbon monoxide rapidly (Schoberth, personal communication). A. woodii contains corrinoids, suggesting that a corrinoid enzyme may also be involved in acetate formation from CO2. Cultures of C. acidi-urici and C. cylindrosporum growing on hypoxanthine reduce CO2 to acetate. Serine and glycine rather than methyl B12 or a methylcorrinoid enzyme complex are considered to be intermediates in the two purine-fermenting anaerobes (41, 42). The finding that these organisms do not oxidize carbon monoxide thus cannot be taken as evidence that carbon monoxide oxidation in C. thermoaceticum and C. formicoaceticum is not catalyzed by one of the enzymes involved in CO2 reduction to acetate.

Growing cultures of C. pasteurianum have been shown to oxidize carbon monoxide slowly (0.001 jimol min-' mg of wet cells ') (16). The electron acceptor for CO oxidation in the growing bacteria is not known. Cell extracts of the organism catalyze the oxidation of carbon monoxide with methyl viologen at the specific rate of 0.025 timol min-' mg of protein at pH 7.2 (37). The enzyme mediating the reaction is reversibly inactivated by both alkyl halides and cyanide (37). C. pasteurianum is not known to reduce CO, to acetate. ACKNOWLEDGMENTS

'IThis work was supported by a grant from the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesherg, and bh the Fonds der Chemischen Industrie.

605

LITERATURE CITED 1. Andreesen, J. R., G. Gottschalk, and H. G. Schlegel. 1970. Clostridium formicoaceticum nov. spec. Isolation, description, and distinction from C. aceticum and C. thermoaceticum. Arch. Microbiol. 72:154-174. 2. Balch, W. E., S. Schoberth, R. S. Tanner, and R. S. Wolfe. 1977. Acetobacterium, a new genus of hydrogenoxidizing, carbon dioxide-reducing anaerobic bacteria. Int. ,J. Syst. Bacteriol. 27:355-361. 3. Barker, H. A. 1967. Biochemical functions of corrinoid compounds. Biochem. J. 105:1-15. 4. Bray, G. A. 1960. A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Anal. Biochem. 1:279-285. 5. Brot, N., and H. Weissbach. 1965. Enzymatic synthesis of methionine. J. Biol. Chem. 240:3064-3070. 6. Cleland, W. W. 1970. Steady state kinetics, p. 1-65. In P. D. Boyer (ed.), The enzymes, vol. 2, 3rd ed. Academic Press Inc., New York. 7. Daniels, L., G. Fuchs, R. K. Thauer, and J. G. Zeikus. 1977. Carbon monoxide oxidation by methanogenic bacteria. J. Bacteriol. 132:118-126. 8. Ferenci, T. 1974. Carbon monoxide-stimulated respiration in methane-utilizing bacteria. FEBS Lett. 41:94-98. 9. Ferenci, T. 1976. The non-growth oxidation of carbon monoxide by Pseudomonas methanica and its relevance to studies of methane oxidation, p. 371-378. In H. G. Schlegel, G. Gottschalk, and N. Pfennig (ed.), Proceedings of the Symposium on Microbial Production and Utilization of Gases (H2, CH4, CO). E. Goltze,

Gottingen. 10. Ferenci, T., T. Str0m, and J. R. Quayle. 1975. Oxidation of carbon monoxide and methane by Pseudomonas methanica. J. Gen. Microbiol. 91:79-91. 11. Firth, R. A., H. A. Hill, J. M. Pratt, R. G. Thorp, and R. J. Williams. 1969. The chemistry of vitamin B12. Some further formation constants. J. Chem. Soc. A 1969:381-386. 12. Fontaine, F., W. H. Peterson, E. McCoy, M. J. Johnson, and G. Ritter. 1942. A new type of glucose fermentation by Clostridium thermoaceticum. J. Bacteriol. 43:701-715. 13. Friedrich, W. 1970. Uber die Reaktion von Kohlenmolnoxid mit Corrinoiden. Z. Naturforsch. Teil B 25:1431-1434. 14. Friedrich, W. 1975. Vitamin B12 und verwandte Corrinoide. Georg Thieme Verlag, Stuttgart. 15. Friedrich, W., and M. Moskophidis. 1971. Weitere Beobachtungen zur Reaktion von Corrinoiden mit Kohlenmonoxid und Chlorkohlensaureestern. Z. Naturforsch. Teil B 26:879-887. 16. Fuchs, G., U. Schnitker, and R. K. Thauer. 1974. Carbon monoxide oxidation by growing cultures of Closti-idium pasteurianium. Eur. J. Biochem. 49:111-115. 17. Ghambeer, R. K., H. G. Wood, M. Schulman, and L. G. Ljungdahl. 1971. Total synthesis of acetate from CO-. III. Inhibition by alkylhalides of the synthesis from CO2, methyltetrahydrofolate, and methyl-BI2 by Clostridium thermoaceticum. Arch. Biochem. Biophvs. 143:471-484. 18. Gottwald, M., J. R. Andreesen, J. LeGall, and L. G. Ljungdahl. 1975. Presence of cytochrome and menaquinone in Clostridium formicoaceticum and Clostridoinm thermoaceticum. .J. Bacteriol. 122:325-328. 19. Herbert, D., P. J. Phipps, and R. E. Strange. 1971. Chemical analysis of microbial cells, p. 209-344. In .J. R. Norris and 1). W. Ribbons (ed.), Methods in microbiology, vol. 5h. Academic Press Inc., New York. 2(1. Hirsch, P. 1968. Plhotosynthetic bacterium growing under carbon monoxide. Nature (London) 217:555-556. 21. Kluyver, A. J., and C. G. Schnellen. 1947. On the

606

22.

23. 24. 25.

26.

27. 28.

29.

30.

31.

32. 33. 34.

DIEKERT ANI) THALJER

fermentation of carbon monoxide by pure CUltures of methane bacteria. Arch. Biochem. 14:57-70. Lang, E., and H. Lang. 1972. Spezifische Farbreaktion zum direkten Nachweis der AmeisensauLre. Z. Anal. Chem. 260:8-10. Ljungdahl, L. G., and H. G. Wood, 1969. 'I'otal synthesis of acetate from CO, by heterotrophic bacteria. Annu. Rev. Microbiol. 23:515-538. Postgate, J. 1970. Carbon monoxide as a basis for primitive life on other planets: a comment. Nature (London) 226:978. Schoberth, S. 1977. Acetic acid from H, and CO2. Formation of acetate by cell extracts of Acetohacterium uwoodii. Arch. Microbiol. 114:143-148. Schonheit, P., C. Wascher, and R. K. Thauer. 1978. A rapid procedure for the purification of ferredoxin from clostridia using polyethyleneimine. FEBS Lett. 89:219-222. Schrauzer, G. N., and L. P. Lee. 1970. The reduction of vitamin B12, by carbon monoxide. Arch. Biochem. Biophys. 138:16-25. Schrauzer, G. N., and W. J. Michaely. 1972. Uber die Methylenblau-Hemmung der Reduktion des Hydroxocobalamins durch Kohlenoxid. Z. Naturforsch. Ted B 27:577-578. Schrauzer, G. N., and J. W. Sibert. 1969. Electron transfer reactions catalyzed by vitamin B12 and related compounds: the reduction of dyes and of riboflavin by thiols. Arch. Biochem. Biophys. 130:257-266. Schulman, M., R. K. Ghambeer, L. G. Ljungdahl, and H. G. Wood. 1973. Total synthesis of acetate from CO,2. VII. Evidence with Clostridium thermoaceticum that the carboxyl of acetate is derived from the carboxvl of pyruvate by transcarboxylation and not by fixation of CO2. J. Biol. Chem. 248:6255-6261. Schulman, M., D. J. Parker, L. G. Ljungdahl, and H. G. Wood. 1972. Total synthesis of acetate from CO2. V. Determination by mass analysis of the different types of acetate formed from "4C02 by heterotrophic bacteria. J. Bacteriol. 109:633-644. Stephen, H., and T. Stephen. 1963. Solubilities of inorganic and organic compounds, vol. 1, part 1. Pergamon Press, Oxford. Stephenson, M. 1949. Bacterial metabolism, 3rd ed., p. 269-270. Longmans and Green, London. Szarkowaka, L., and M. Klingenberg. 1963. On the role of ubiquinone in mitochondria (spectrophotometric

J. BAC'TF,RIOL,.

35.

36.

37.

38. 39.

40.

41.

42.

43. 44. 45.

46.

47.

and chemical measurements of its redox reactions). Biochem. Z. 338:674-697. Taylor, R. T., and H. Weissbach. 1967. N'-methyltetrahydrofolate-homocysteine transmethylase. Propylation characteristics with the use of a chenmical reducing system and purified enzyme. J. Biol. Chem. 242:1509-1516. Taylor, R. T., C. Whitfield, and H. Weissbach. 1968. Chemical propy,lation of vitamin B2, transmethylase: anomalous behavior of S-adenosyl-l-methionine. Arch. Biochem. Biophys. 125:240-252. Thauer, R. K., G. Fuchs, B. Kaufer, and U. Schnitker. 1974. Carbon monoxide oxidation in cell-free extracts of Clostridium pasteurianum. Eur. J. Biochem. 45:343-349. Thauer, R. K., K. Jungermann, and K. Decker. 1977. Energy conservation in chemotrophic anaerobic bacteria. Bacteriol. Rev. 41:1003-180. Thauer, R. K., E. Rupprecht, and K. Jungermann. 1970. Separation of 4C-formate from CO2 fixation metabolites by isoionic-exchange chromatography. Anal. Biochem. 38:461-468. Uffen, R. L. 1976. Anaerobic growth of a Rhodopseudomonas species in the dark with carbon monoxide as sole carbon and energy substrate. Proc. Natl. Acad. Sci. U.S.A. 73:3298-3302. Vogels, G. D., and C. van der Drift. 1976. Degradation of purines and pyrimidines by microorganisms. Bacteriol. Rev. 40:403-468. Wagner, R., and J. R. Andreesen. 1977 Differentiation between Clostridium acidiurici and Clostridium cylindrosporum on the basis of specific metal requirements for formate dehydrogenase formation. Arch. Microbiol. 114:219-224. Weissbach, H., and R. T. Taylor. 1970. Role of vitamin B12 and folic acid in methionine synthesis. Vitam. Horm. (N.Y.) 28:415-440. Wood, J. M., and R. S. Wolfe. 1966. Propvlation and purification of a B12 enzv-me involved in methane formation. Biochemistry 5:3598-3603. Yagi, T. 1958. Enz-mic oxidation of carbon monoxide. Biochim. Biophys. Acta 30:194-195. Yagi, T. 1959. Enzvmic oxidation of carbon monoxide. J. Biochem. (Tokyo) 46:949-955. Yagi, T., and N. Tamiya. 1962. Enzymic oxidation of carbon monoxide. Biochim. Biophys. Acta 65:508-509.